Reaching ultimate diffraction-limit spatial resolution, which is approximately half a wavelength of a light source, is very important technology with imaging a target object embedded deep within scattering media, such as biological tissues. Multiple scattering events attenuate light waves that preserve original incidence momenta and generate multiply scattered waves, which act as strong background noise. As target depth is increased, these combined effects lead to an exponential decrease of a signal to noise ratio (SNR). Because of this, sub-micron scales of important biological reactions occurring inside living tissues have been out of reach as a consequence, and optical microscopy was unable to effectively support an investigation of the early stages of disease progression and the study of nervous systems.
When considering a target spatial resolution close to the ultimate diffraction limit, the attenuation of SNR by the multiple light scattering is not the only problem. In fact, a so-called specimen-induced aberration is an equally important issue to address. A signal wave that preserves original incidence momenta is not only attenuated in its intensity by the multiple light scattering, but its phase is also retarded due to the heterogeneity of the medium.
These phase retardations of the signal wave vary depending on a propagation angle, and the phase retardations take place for both an incident path and a returning path. These angle-dependent phase retardations cause a distortion of a reconstructed object image, and make them the main source of specimen-induced aberration. Also, they also hinder a proper accumulation of the signal wave in the image reconstruction stage and cause a further reduction in SNR in addition to that caused by multiple light scattering.
For example, the specimen-induced aberration of the typical biological tissue with thicknesses of a few scattering mean free paths (MFPs) can attenuate the single scattering intensity of the target object image by hundreds of times. This detrimental aberration effect is much more pronounced for high-resolution imaging, as waves propagating at large incidence angle retaining high-spatial frequency information tend to pass through effectively longer paths and are thus more likely to experience large phase retardation. The real challenge of these aberrations when imaging the target object in scattering media is that they are extremely difficult to identify in the presence of strong multiple light scattering.
In this regard, numerous attempts have tried to deal with either scattering or aberration individually in the past researches. The method for dealing with scattering uses a temporal gating and/or a confocal gating for the selective collection of a single-scattered wave. However, the existence of the specimen-induced aberration easily undermines these gating operations.
Using an eigenchannel to better accumulate the signal wave has been attempted, in paper of Popoff, etc. “Exploiting the Time-Reversal Operator for Adaptive Optics, Selective Focusing, and Scattering Pattern Analysis (Physical Review Letters 107, 263901 (2011))”, but does not guarantee aberration compensation.
In Korean Patent number 10-1688873 “OPTICAL COHERENCE TOMOGRAPHY” which is applied by inventors of a present invention and a paper “Imaging deep within a scattering medium using collective accumulation of single-scattered waves (Nature Photonics 9, 253-258, 2015)”, a method termed collective accumulation of single scattering (CASS) microscopy was proposed.
The CASS method combines both time-gated detection and spatial input-output correlation. The CASS method was used to preferentially accumulate the single-scattered wave, which is the wave scattered only once by the target object, but not at all by the medium. This has resulted in a dramatic increase of working depth such that spatial resolution of 1.5 μm can be maintained up to 11 MFPs.
For example, if the conventional method is used, the spatial resolution of 0.6 μm can be maintained only up to 8 MFPs at the same condition. However, the specimen-induced aberration in the biological tissue hinders the accumulation of the single-scattered wave. Strictly speaking, the achievable depth is even shallower than this fundamental limit by a few MFPs.
On the other hand, the method for dealing with aberration has been actively proposed in the field of adaptive optics. The aberration used to be characterized on the basis of Zernike polynomials by direct wavefront sensing or experimental feedback control. These approaches have been particularly useful for fluorescence imaging because only the aberration correction of the incident wave matters. Nevertheless, the ability to address both multiple scattering and aberrations has been limited by an insufficient number of control elements in a wavefront shaping device. The adaptive optics for the coherent imaging has proved even more difficult to implement when multiple scattering noise exists, and successful implementations have only been reported for cases with negligible multiple light scattering.
If the background noise caused by multiple light scattering is not addressed, the intensity of the single-scattered wave is less than the intensity of the background noise caused by the multiple-scattered wave. On the other hand, if aberration is not addressed, then the single-scattered wave is accumulated so ineffectively that they may not effectively compete with the multiple-scattered wave.